A siRNA targeting glioblastoma and its application

Abstract

The present invention relates to a siRNA targeting glioblastoma and its application, belonging to the field of biotechnology. The present invention provides a siRNA targeting glioblastoma, the antisense strand of the siRNA can specifically bind to the target nucleic acid to trigger the degradation of the target nucleic acid, wherein the target nucleic acid includes the pathogenic gene of glioblastoma, and the pathogenic gene of glioblastoma includes the gene encoding ribonucleotide reductase subunit M2 and / or the gene encoding heat shock protein 47. Studies have shown that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 are significantly correlated with the survival of glioblastoma patients. These two genes can be used as therapeutic targets for glioblastoma, and siRNA that can effectively knock down the expression of these two genes in glioblastoma patients has great application prospects in the preparation of drugs for preventing and / or treating glioblastoma.

Description

Technical Field

[0001] The invention relates to siRNA targeting glioblastoma and application thereof, belonging to the field of biotechnology. Background Art

[0002] Glioma is one of the most common brain tumors, accounting for 30% of brain tumors in the central nervous system. According to the grading standards of the World Health Organization (WHO), gliomas are divided into grades I to IV. Among them, the most malignant grade IV glioma is also called glioblastoma multiforme (GBM). Glioblastoma often occurs in the brain parenchyma and is diffusely dispersed. It is the most common and most invasive type of glioma in clinical practice. The median survival time of patients is only 12 to 18 months, and the 5-year survival rate is less than 5%.

[0003] At present, the standard treatments for glioblastoma mainly include surgical resection, radiotherapy (RT) and chemotherapy. Among them, the only first-line drug for chemotherapy is temozolomide (TMZ). However, it was found in the clinical treatment process that about 55% of glioblastoma patients are primary resistant to temozolomide, and another part of glioblastoma patients will inevitably acquire a temozolomide-resistant phenotype during temozolomide treatment, resulting in poor treatment effect. Therefore, more effective therapeutic drugs for glioblastoma are urgently needed.

[0004] With the advancement of next-generation sequencing technology, people have a deeper understanding of the molecular basis and genomic picture of glioblastoma, and many researchers have begun to try targeted treatment of glioblastoma. However, due to the existence of the blood-brain barrier and the high heterogeneity of glioblastoma, many glioblastoma targeted therapeutic drugs in the clinical research stage have poor therapeutic effects, and no significant progress has been made in extending patients' overall survival and improving prognosis. Therefore, there is an urgent need to break through the efficiency of central drug delivery and tumor heterogeneity, so as to obtain glioblastoma targeted therapeutic drugs with better therapeutic effects.

[0005] RNA interference (RNAi) is a specific gene silencing phenomenon mediated by small interfering RNA (siRNA) and involving specific enzymes that has been discovered in recent years and is ubiquitous in organisms. siRNA is a double-stranded RNA molecule composed of 20 to 25 nucleotides. In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation and inhibits the expression of specific genes. In theory, RNAi can silence all genes. Therefore, siRNA can be used as a targeted therapeutic drug to specifically regulate the expression of disease-related genes.

[0006] If the expression of glioblastoma-related genes in vivo can be effectively knocked down by siRNA, it will be very helpful for the targeted treatment of glioblastoma. In addition, since siRNA is specific and effective in knocking down disease-related genes, the development of glioblastoma targeted therapeutic drugs based on RNAi is expected to break through the central administration efficiency and tumor heterogeneity. However, naked siRNA is extremely susceptible to nuclease degradation, which makes the success of RNAi therapy largely depend on siRNA carriers and delivery methods. Traditional siRNA delivery systems, such as lipid nanoparticles, cationic polymers and viruses, have problems such as easy biodegradation, low biocompatibility, insufficient circulatory stability and weak targeting ability. Therefore, on the basis of developing siRNA that can effectively knock down the expression of glioblastoma-related genes in vivo, it is also critical to find a delivery system that can effectively deliver siRNA into the nerve center, and can maintain the stability and biological activity of siRNA in vivo for a long time, and at the same time, it has high biocompatibility. Targeted treatment of glioblastoma is also critical. Summary of the invention

[0007] To solve the above problems, the present invention provides a siRNA targeting glioblastoma, wherein the siRNA comprises a sense chain and an antisense chain; the sense chain of the siRNA can be at least partially reverse complementary to the antisense chain to form a double-stranded region; the antisense chain of the siRNA can specifically bind to a target nucleic acid through base complementary pairing to induce degradation of the target nucleic acid; the target nucleic acid includes a pathogenic gene of glioblastoma; the pathogenic gene of glioblastoma includes a gene encoding ribonucleotide reductase subunit M2 (RRM2 gene) and / or a gene encoding heat shock protein 47 (HSP47) (Serpinh1 gene).

[0008] In one embodiment of the present invention, the target nucleic acid includes mRNA encoding ribonucleotide reductase subunit M2 and / or mRNA encoding heat shock protein 47.

[0009] In one embodiment of the present invention, the pathogenic genes of glioblastoma further include a gene encoding epidermal growth factor receptor (EGFR gene), miR-214 and / or a gene encoding vascular endothelial growth factor receptor (VEGFR gene).

[0010] In one embodiment of the present invention, the target nucleic acid includes mRNA encoding epidermal growth factor receptor, miR-214 and / or mRNA encoding vascular endothelial growth factor receptor.

[0011] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.9;

[0012] When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.5, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.10.

[0013] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.9;

[0014] When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.10.

[0015] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.6;

[0016] When the pathogenic gene of glioblastoma is miR-214, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.7;

[0017] When the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.8.

[0018] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.6;

[0019] When the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.7;

[0020] When the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.8.

[0021] The present invention also provides a recombinant nucleic acid molecule, which comprises shRNA; the shRNA comprises the sense strand of the siRNA and the antisense strand of the siRNA; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

[0022] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.14;

[0023] When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.15.

[0024] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.14;

[0025] When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.15.

[0026] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.11;

[0027] When the pathogenic gene of glioblastoma is miR-214, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.12;

[0028] When the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.13.

[0029] In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.11;

[0030] When the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.12;

[0031] When the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the nucleotide sequence of the shRNA is shown as SEQ ID NO.13.

[0032] In one embodiment of the present invention, the recombinant nucleic acid molecule further comprises a targeting nucleic acid molecule, a promoter and / or a functional nucleic acid molecule; the targeting nucleic acid molecule encodes a neural center targeting peptide; and the functional nucleic acid molecule encodes a lysosome-related membrane protein.

[0033] In one embodiment of the present invention, the neural center targeting peptide includes a rabies virus glycoprotein peptide (RVG) that can penetrate the blood-brain barrier, a glioma targeting peptide (Angiopep-2), apolipoprotein (APOE) and / or a cell penetrating peptide (CPP).

[0034] In one embodiment of the present invention, the promoter includes a cytomegalovirus promoter (CMV promoter, CMV Promoter), a lentivirus promoter (EF1 promoter, EF1 Promoter) and / or a polymerase III promoter (U6 promoter).

[0035] In one embodiment of the present invention, the promoter is a cytomegalovirus promoter; the nucleotide sequence of the cytomegalovirus promoter is shown in SEQ ID NO.16.

[0036] In one embodiment of the present invention, the lysosome-associated membrane protein includes lysosome-associated membrane protein 2B (LAMP2B), transmembrane protein CD63, transmembrane protein CD9 and / or transmembrane protein CD81.

[0037] In one embodiment of the present invention, the targeting nucleic acid molecule encodes a rabies virus glycoprotein peptide (RVG); the functional nucleic acid molecule encodes a lysosome-associated membrane protein 2B; the targeting nucleic acid molecule is inserted between functional nucleic acid molecules to form a chimeric fragment; the nucleotide sequence of the chimeric fragment is shown in SEQ ID NO.17.

[0038] In one embodiment of the present invention, the recombinant nucleic acid molecule includes a cytomegalovirus promoter, a chimeric fragment and an shRNA connecting fragment connected in series in sequence; the shRNA connecting fragment includes an shRNA corresponding to an siRNA targeting an mRNA encoding epidermal growth factor receptor, an shRNA corresponding to an siRNA targeting an mRNA encoding vascular endothelial growth factor receptor, an shRNA corresponding to an siRNA targeting an mRNA encoding ribonucleotide reductase subunit M2, an shRNA corresponding to an siRNA targeting an mRNA encoding heat shock protein 47, and an shRNA corresponding to an siRNA targeting miR-214, which are connected in series in sequence.

[0039] In one embodiment of the present invention, the nucleotide sequence of the shRNA linker fragment is shown as SEQ ID NO.18.

[0040] The present invention also provides a recombinant plasmid, wherein the recombinant plasmid expresses the above siRNA; or the recombinant plasmid carries the above recombinant nucleic acid molecule.

[0041] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.

[0042] In one embodiment of the present invention, the plasmid vector comprises pcDNA6.2 plasmid, pLKO.1 plasmid, PGEM-3zf plasmid, PUC19 plasmid and / or PUC57 plasmid.

[0043] In one embodiment of the present invention, the method for preparing the recombinant plasmid is: the above-mentioned recombinant nucleic acid molecule is digested with a linearized vector and then connected to obtain the recombinant plasmid.

[0044] The present invention also provides an engineered cell, wherein the genome of the engineered cell is integrated with the above siRNA; or, the genome of the engineered cell is integrated with the above recombinant nucleic acid molecule; or, the engineered cell carries the above recombinant plasmid.

[0045] In one embodiment of the invention, the engineered cells include liver cells.

[0046] In one embodiment of the present invention, the engineered cells are transformed liver cells of the body.

[0047] The present invention also provides a small extracellular vesicle, wherein the small extracellular vesicle encapsulates the above-mentioned siRNA; or, the small extracellular vesicle encapsulates the above-mentioned recombinant nucleic acid molecule; or, the small extracellular vesicle encapsulates the above-mentioned recombinant plasmid; or, the small extracellular vesicle is secreted by the above-mentioned engineered cells.

[0048] In one embodiment of the present invention, the small extracellular vesicles secreted by the engineered cells are packaged with siRNA; the siRNA is obtained by expressing the recombinant nucleic acid molecule in the engineered cells.

[0049] The present invention also provides the use of the above siRNA or the above recombinant nucleic acid molecule or the above recombinant plasmid or the above engineered cell or the above small extracellular vesicle in the preparation of a drug for preventing and / or treating brain tumors in the central nervous system.

[0050] In one embodiment of the present invention, the composition of the drug comprises an inhibitor; the inhibitor comprises the above-mentioned siRNA, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned engineered cell and / or the above-mentioned small extracellular vesicle.

[0051] In one embodiment of the present invention, the inhibitor is the above-mentioned engineered cell.

[0052] In one embodiment of the present invention, the composition of the drug further comprises a pharmaceutically acceptable excipient.

[0053] In one embodiment of the present invention, the pharmaceutically acceptable excipient further includes a solvent.

[0054] In one embodiment of the present invention, the central nervous system brain tumor includes glioma; the glioma includes glioblastoma.

[0055] The present invention also provides a drug for preventing and / or treating brain tumors in the central nervous system, wherein the drug comprises an inhibitor; the inhibitor comprises the above-mentioned siRNA, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned engineered cells and / or the above-mentioned small extracellular vesicles.

[0056] In one embodiment of the present invention, the inhibitor is the above-mentioned engineered cell.

[0057] In one embodiment of the present invention, the composition of the drug further comprises a pharmaceutically acceptable excipient.

[0058] In one embodiment of the present invention, the pharmaceutically acceptable excipient further includes a solvent.

[0059] In one embodiment of the present invention, the central nervous system brain tumor includes glioma; the glioma includes glioblastoma.

[0060] The technical solution of the present invention has the following advantages:

[0061] 1. The present invention provides a siRNA targeting glioblastoma, wherein the antisense strand of the siRNA can specifically bind to a target nucleic acid through complementary base pairing to induce degradation of the target nucleic acid; the target nucleic acid includes a pathogenic gene of glioblastoma; the pathogenic gene of glioblastoma includes a gene encoding ribonucleotide reductase subunit M2 (RRM2 gene) and / or a gene encoding heat shock protein 47 (HSP47) (Serpinh1 gene). Through differential gene enrichment analysis and gene pathway enrichment analysis between glioblastoma patients and healthy people, it was found that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 were significantly correlated with the survival of glioblastoma patients. It can be seen that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 can be used as therapeutic targets for glioblastoma. Therefore, siRNA that can effectively knock down the expression of these two genes in glioblastoma patients has great application prospects in the preparation of drugs for the prevention and / or treatment of central nervous system brain tumors, especially glioblastoma.

[0062] Furthermore, the pathogenic genes of glioblastoma also include genes encoding epidermal growth factor receptor (EGFR gene), miR-214 and / or genes encoding vascular endothelial growth factor receptor (VEGFR gene). The genes encoding epidermal growth factor receptor, miR-214 and genes encoding vascular endothelial growth factor receptor are also significantly correlated with the survival of glioblastoma patients. It can be seen that the genes encoding epidermal growth factor receptor, miR-214 and genes encoding vascular endothelial growth factor receptor can also be used as therapeutic targets for glioblastoma. Therefore, siRNA that can effectively knock down the expression of these three genes in glioblastoma patients has great application prospects in the preparation of drugs for preventing and / or treating central nervous system brain tumors, especially glioblastoma.

[0063] Further, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense chain of the siRNA is shown as SEQ ID NO.4, and the nucleotide sequence of the antisense chain is shown as SEQ ID NO.9; when the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the sense chain of the siRNA is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense chain is shown as SEQ ID NO.10; when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the nucleotide sequence of the sense chain of the siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense chain is shown as SEQ ID NO.6; when the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the sense chain of the siRNA is shown as SEQ ID NO.2, and the nucleotide sequence of the antisense chain is shown as SEQ ID NO. NO.7; when the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8. The siRNA can target the pathogenic gene of glioblastoma; studies have shown that the interference efficiency of the siRNA is high and can significantly knock down the expression of the pathogenic gene of glioblastoma in vivo; and studies have shown that the small extracellular vesicles encapsulating the siRNA can significantly inhibit the growth of glioblastoma in the mouse brain. Therefore, the siRNA has great application prospects in the preparation of drugs for preventing and / or treating central nervous system brain tumors, especially glioblastoma.

[0064] 2. The present invention also provides a recombinant nucleic acid molecule, which comprises shRNA, a targeting nucleic acid molecule, a promoter and / or a functional nucleic acid molecule; the shRNA comprises a sense strand of a siRNA targeting glioblastoma and an antisense strand of a siRNA targeting glioblastoma; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure; the targeting nucleic acid molecule encodes a neural center targeting peptide; and the functional nucleic acid molecule encodes a lysosome-related membrane protein. The present invention uses a synthetic biology method to connect a neural center targeting peptide that can penetrate the blood-brain barrier, a lysosome-related membrane protein that can form small extracellular vesicles, and an shRNA containing siRNA in series to obtain a gene loop; the gene loop is injected into the body by intravenous injection, and the body's own liver can be used as a biological reaction base, and small extracellular vesicles encapsulating therapeutic siRNA can be self-assembled in vivo to achieve precise delivery and multi-target combined treatment of glioblastoma RNAi drugs. Therefore, the recombinant nucleic acid molecule has great application prospects in the preparation of drugs for preventing and / or treating central nervous system brain tumors, especially glioblastoma.

[0065] 3. The present invention provides a small extracellular vesicle, which encapsulates siRNA targeting glioblastoma. Small extracellular vesicles (sEV) are small membrane particles (with a particle size of 30 to 150 nanometers) secreted by cells. As an important means of cell communication, they help cells to complete communication activities by transporting nucleic acids and proteins between neighboring cells. Compared with traditional delivery systems, small extracellular vesicles can escape phagocytosis, prolong the half-life of the drug in vivo, and reduce immunogenicity due to their natural properties. Delivery of siRNA targeting glioblastoma by small extracellular vesicles has the following advantages: first, siRNA targeting glioblastoma can be effectively delivered into the nerve center, and the stability and biological activity of siRNA targeting glioblastoma can be maintained for a long time in vivo, and at the same time, it also has high biocompatibility, which helps siRNA targeting glioblastoma to inhibit the expression of glioblastoma pathogenic genes in vivo, thereby exerting an anti-tumor effect; second, siRNA targeting glioblastoma can be delivered by small extracellular vesicles, which can effectively avoid the problems faced by delivery of extracellular vesicles derived from exogenous cells, such as complex technical operations, expensive costs, and endotoxins; third, experiments have shown that the injection of the small extracellular vesicles of the present invention into mice can significantly inhibit the growth of glioblastoma in the mouse brain, and the targeted treatment of glioblastoma is significant. Therefore, the small extracellular vesicles are very promising in the preparation of drugs for preventing and / or treating central nervous system brain tumors, especially glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 : Association of the gene encoding deoxyribonucleotide reductase (RRM2) with patient survival.

[0067] Figure 2 : Association of the gene encoding heat shock protein 47 (HSP47) with patient survival.

[0068] Figure 3 : Interference efficiency of siRNA targeting VEGFR gene.

[0069] Figure 4 : Interference efficiency of siRNA targeting RRM2 gene.

[0070] Figure 5 : Interference efficiency of siRNA targeting Serpinh1 gene.

[0071] Figure 6 : Construction map of a multi-target gene loop with five siRNAs in series.

[0072] Figure 7 : Recombinant plasmid CMV-RVG-siR EGFR+VEGFR+RRM2+Serpinh1+anti-214 Plasmid map.

[0073] Figure 8 : Experimental flow chart for in vitro functional validation of self-assembled sEV-siRNA.

[0074] Fig. 9 : Effect of self-assembled sEV-siRNA on EGFR gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0075] Fig.10 : Effect of self-assembled sEV-siRNA on VEGFR gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0076] Fig.11 : Effect of self-assembled sEV-siRNA on RRM2 gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0077] Fig.12 : Effect of self-assembled sEV-siRNA on Serpinh1 gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0078] Fig.13 : Effects of self-assembled sEV-siRNA on proteins (epidermal growth factor receptor, vascular endothelial growth factor, deoxyribonucleotide reductase and heat shock protein 47) (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target proteins).

[0079] Fig.14 : Small animal in vivo imaging results of different groups of mice.

[0080] Fig.15 :Therapeutic effect of gene circuit in mice (tail vein injection of gene circuit inhibits the growth of orthotopic xenograft intracranial tumors in mice). DETAILED DESCRIPTION

[0081] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0082] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0083] Experimental Example 1: Acquisition of therapeutic targets for glioblastoma

[0084] The specific process is as follows:

[0085] According to the characteristics of glioblastoma, such as rapid proliferation, strong angiogenesis, and immune escape, epidermal growth factor receptor (EGFR), miR-214, and vascular endothelial growth factor (VEGFR) were first selected as possible therapeutic targets for GBM, and then the TCGA database was used to screen for therapeutic targets unique to glioblastoma. The specific screening process was as follows: the gene information of 10 healthy people and 528 glioblastoma patients was downloaded from the Affymetrix HG-UG133A platform, and the genes with significant changes in glioblastoma patients were found through differential gene enrichment analysis and gene pathway enrichment analysis. These genes were then associated with the survival of patients. The results are shown in Figure 2. Figure 1~Figure 2 .Depend on Figure 1~Figure 2 It can be seen that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 are both significantly correlated with the survival of glioblastoma patients. Therefore, the new therapeutic targets for glioblastoma, deoxyribonucleotide reductase (RRM2) and heat shock protein 47 (HSP47, encoded by the Serpinh1 gene), were finally identified.

[0086] Experimental Example 2: Acquisition of siRNA targeting glioblastoma

[0087] The specific process is as follows:

[0088] 1. Design

[0089] Taking mRNA encoding epidermal growth factor receptor (EGFR), miR-214, mRNA encoding vascular endothelial growth factor receptor (VEGFR), mRNA encoding ribonucleotide reductase subunit M2 (RRM2) and mRNA encoding heat shock protein 47 (HSP47, encoded by Serpinh1 gene) as target genes, siRNAs targeting EGFR gene, miR-214 gene, VEGFR gene, RRM2 gene and Serpinh1 gene were designed respectively (see Table 1 for details, Table 1 is the complementary DNA sequence of the siRNA sense chain and antisense chain), and the siRNAs are as follows:

[0090] siRNA targeting EGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.6, which is named EGFR-siRNA;

[0091] siRNA targeting miR-214: the nucleotide sequence of the sense strand is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7, which is named miR-214-siRNA;

[0092] siRNA1 targeting VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.25, which is named VEGFR-siRNA1;

[0093] siRNA2 targeting VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8, which is named VEGFR-siRNA2;

[0094] siRNA3 targeting VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.20, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.26, which is named VEGFR-siRNA3;

[0095] siRNA1 targeting the RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.21, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.27, which is named RRM2-siRNA1;

[0096] siRNA2 targeting RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.9, which is named RRM2-siRNA2;

[0097] siRNA3 targeting RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.22, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.28, which is named RRM2-siRNA3;

[0098] siRNA1 targeting Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.29, which is named Serpinh1-siRNA1;

[0099] siRNA2 targeting Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.24, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.30, which is named Serpinh1-siRNA2;

[0100] siRNA3 targeting Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.10, which is named Serpinh1-siRNA3;

[0101] 2. Screening

[0102] 5 μL of transfection reagent lipo2000 (purchased from Thermo Fisher Scientific) was mixed with 100 μL of Opti-MEM (purchased from Thermo Fisher Scientific) and allowed to stand for 5 min to obtain mixture A; different siRNAs were diluted to a concentration of 100 pmol with 100 μL of Opti-MEM and allowed to stand for 5 min to obtain mixture B; mixture A and mixture B were mixed and allowed to stand for 20 min to obtain transfection solutions containing different siRNAs;

[0103] U87 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured at 1×10 5 The cells were inoculated at an inoculum size of 100 μg / well into a 6-well plate with 2 mL of DMEM medium (purchased from Thermo Fisher Scientific) containing 10% (v / v) fetal bovine serum added to each well, and cultured in a cell culture incubator with 5% (v / v) CO2 and 37°C for 24 h. After 24 h of culture, a blank control group, a negative control group (siNC), an EGFR-siRNA experimental group, a miR-214-siRNA experimental group, a VEGFR-siRNA1 experimental group, a VEGFR-siRNA2 experimental group, a VEGFR-siRNA3 experimental group, a RRM2-siRNA1 experimental group, a RRM2-siRNA2 experimental group, a RRM2-siRNA3 experimental group, a Serpinh1-siRNA1 experimental group, a Serpinh1-siRNA2 experimental group, and a Serpinh1-siRNA3 experimental group were set up in the 6-well plate, with 4 replicate wells in each group.

[0104] After the setup was completed, transfection solutions containing different siRNAs were added to the wells of the experimental groups (the EGFR-siRNA experimental group was given a transfection solution containing siRNA targeting the EGFR gene, the miR-214-siRNA experimental group was given a transfection solution containing siRNA targeting the miR-214 gene, the VEGFR-siRNA1 experimental group was given a transfection solution containing siRNA1 targeting the VEGFR gene, and so on), and transfection solutions containing siNC were added to the wells of the negative control group (the nucleotide sequence of the positive chain of siNC is shown in SEQ ID NO.31, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.32), and transfection solutions without siRNA were added to the blank control group, and the cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 6 h for transfection; after 6 h of transfection, the old cell culture medium was discarded, and 2 mL of new DMEM culture medium containing 10% (v / v) fetal bovine serum was added to each well of the 6-well plate, and the cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 24 h. h for transfection;

[0105] 24 h after transfection, cell lysate (purchased from Sigma) was added to a 6-well plate at a volume of 500 μL per well, and chloroform was added to a 6-well plate at a volume of 100 μL per well, first vortexed vigorously, then placed on ice for 5 min, and finally centrifuged at 12000 rpm and 4°C for 10 min; after centrifugation, the supernatant was taken, and isopropanol of the same volume as the supernatant was added, first placed at room temperature (25°C) for 16 h, and then centrifuged at 12000 rpm and 4°C for 10 min; after centrifugation, the precipitate was taken, and the precipitate was resuspended to the original volume (i.e., the same volume as the supernatant) with 75% (v / v) ethanol, and centrifuged at 12000 rpm and 4°C for 10 min; after centrifugation, the precipitate was taken to obtain mRNA; the extracted mRNA was used as the sample to be tested, and 18S was used as the internal standard gene to detect the expression of the target gene in the cells after different siRNA interference by qPCR (C was obtained by qPCR). T The relative levels of mRNA were normalized to 18S rRNA and the The expression level of the target gene was determined by the method) to verify the interference efficiency of the designed siRNA. The verification results are shown in Figure 3~Figure 5 ;

[0106] The specific steps of qPCR are as follows:

[0107] Reverse transcription: Add the reagents according to the reaction system in Table 2 to a 200 μL enzyme-free centrifuge tube, vortex to mix, and place in a PCR instrument to obtain cDNA (see Table 2 for the specific reverse transcription reaction procedure);

[0108] qPCR: Add the reagents according to the reaction system in Table 3 to a 200 μL enzyme-free centrifuge tube, vortex and centrifuge, and then add to a 96-well plate (see Table 3 for the specific qPCR reaction procedure and Table 4 for the primers used for qPCR).

[0109] Depend on Figure 3 It can be seen that among the siRNAs targeting the VEGFR gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of VEGFRmRNA.

[0110] Depend on Figure 4 It can be seen that among the siRNAs targeting the RRM2 gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of RRM2 mRNA.

[0111] Depend on Figure 5 It can be seen that among the siRNAs targeting the Serpinh1 gene, siRNA3 has the best interference efficiency and significantly knocks down the expression of Serpinh1 mRNA.

[0112] Table 1 siRNA and its sequence

[0113]

[0114] Table 2 Reverse transcription reaction system and reaction procedure

[0115]

[0116] Table 3 qPCR reaction system and reaction procedure

[0117]

[0118] Table 4 qPCR primers and their sequences

[0119]

[0120] Experimental Example 3: Effects of self-assembled sEV-siRNA on target proteins and mRNA in vitro

[0121] The specific process is as follows:

[0122] 1. Recombinant plasmid CMV-RVG-siR EGFR+VEGFR+RRM2+Serpinh1+anti-214 Construction

[0123] According to the siRNA targeting the EGFR gene, the nucleotide sequence of the sense strand is shown in SEQ ID NO.1 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.6, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.11), and it is named Hsa-siEGFR;

[0124] According to the siRNA targeting miR-214, the nucleotide sequence of the sense strand is shown in SEQ ID NO.2 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.12), and it is named Anti-miR-214;

[0125] According to the siRNA targeting the VEGFR gene, the nucleotide sequence of the sense strand is shown in SEQ ID NO.3 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.13), and it is named mmu-siVEGFR;

[0126] According to the siRNA targeting the RRM2 gene, the nucleotide sequence of the sense strand is shown in SEQ ID NO.4 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.9, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.14), and it is named Hsa-siRRM2;

[0127] According to the siRNA targeting the Serpinh1 gene, the nucleotide sequence of the sense strand is shown in SEQ ID NO.5 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.10, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.15), and it is named Hsa-siSerpinh1;

[0128] The cytomegalovirus promoter (CMV promoter), the chimeric fragment of the gene encoding the rabies virus glycoprotein peptide (RVG) and the gene encoding the lysosomal associated membrane protein 2B (LAMP2B), Hsa-siEGFR, mmu-siVEGFR, Hsa-siRRM2, Hsa-siSerpinh1 and Anti-miR-214 were sequentially connected in series to obtain a multi-target gene loop (the construction map of the multi-target gene loop of five siRNAs in series is shown in Figure 6 The nucleotide sequence of the cytomegalovirus promoter is shown in SEQ ID NO.16, the nucleotide sequence of the chimeric fragment is shown in SEQ ID NO.17, and the nucleotide sequence of the multi-target gene loop is shown in SEQ ID NO.43).

[0129] The multi-target gene loop was synthesized, and the multi-target gene loop and the PCDNA6.2 plasmid vector (purchased from Shanghai Jima Co., Ltd.) were double-digested with BamHI and XhoI (purchased from Thermo Fisher Scientific) (the digestion system is shown in Table 5, and the digestion reaction was carried out at 37°C for 30 min and then inactivated at 85°C for 5 min). After the digestion reaction was completed, the product was run on gel electrophoresis, and the target fragment was recovered from the gel to obtain the double-digested vector and insert fragment; the double-digested vector and insert fragment were ligated using T4 DNA ligase and T4 DNA ligase buffer (purchased from Takara) (the ligation system is shown in Table 6, and the ligation reaction was incubated at 16°C for 14 h for ligation) to obtain the ligation product; the ligation product was transformed into Escherichia coli competent cells DH5α (purchased from Qingke Biotechnology) to obtain the transformation product; the transformation product was streaked on LB agar plate medium (formula: 5g / L yeast, 10g / L peptone, 10g / L sodium chloride and 18g / L agar powder) containing 50μg / mL ampicillin (Amp), cultured at 37℃ for 12 h, and a single colony was picked; the single colony was inoculated into 3 mL LB liquid medium (formula: 5g / L yeast, 10g / L peptone and 10g / L sodium chloride) containing 50μg / mL spectinomycin, and cultured at 37℃ for 14 h to obtain a bacterial solution; the recombinant plasmid in the bacterial solution was extracted and purified for sequencing, and the recombinant plasmid CMV-RVG-siR was obtained after successful sequencing. EGFR+VEGFR+RRM2+Serpinh1+anti-214 (For the plasmid map of the recombinant plasmid, see Figure 7 , the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.44).

[0130] 2. Effects of self-assembled sEV-siRNA on target proteins and mRNA in vitro

[0131] according to Figure 8 The experimental process was to compare the empty plasmid and the recombinant plasmid CMV-RVG-siR EGFR +VEGFR+RRM2+Serpinh1+anti-214 Six-week-old BALB / c mice (purchased from Jicui Pharmaceutical) were injected with the drug via tail vein at a dose of 10 mg / kg (solvent: 200 μL PBS buffer), twice a day for a total of four injections. Six hours after the last injection, plasma was obtained by removing the eyeballs and bleeding, and exosomes (exosomes are sEV-siRNA produced by self-assembly of multi-target gene loops in mice) were extracted from the plasma of mice using ultracentrifugation. U87 cells (purchased from Shanghai Institute of Cell Biology) were cultured at 7.5×10 6The cells were inoculated into 6-well plates with 2 mL of high-glucose medium (purchased from Gibco) containing 10% (v / v) fetal bovine serum (NoviZan) added to each well, and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 12 h. After 12 h of culture, 100 μL of high-glucose medium containing 2% (v / v) fetal bovine serum containing 120 μg of exosomes was added to the wells in which U87 cells were cultured, and co-incubated in a 5% (v / v) CO2, 37°C cell culture incubator. After 24 h of co-incubation, the expression level of the target gene in the cells was detected by Realtime-PCR. The detection results are shown in Figure 9~Figure 12 After 48 h of co-incubation, the expression level of the target protein (protein encoded by the target gene) in the cells was detected by Western blot. The test results are shown in Fig.13 ; Among them, the Realtime-PCR experimental process is as follows:

[0132] RNA extraction: After adding 1 mL of cell lysis solution (purchased from Takara) to each well of a 6-well plate, place it on ice for 45 minutes and shake it vigorously every 15 minutes to obtain a cell lysis product; the cell lysis product was centrifuged at 12000×g and 4°C for 10 minutes to remove cell residues. After centrifugation, supernatant A was taken; 200 μL of chloroform was added to supernatant A, and it was shaken vigorously first, and then placed on ice to stand for stratification (taking 5 minutes) to obtain an extraction product; the extraction product was centrifuged at 12000×g and 4°C for 15 minutes. After centrifugation, supernatant B was taken; 600 μL of isopropanol was added to supernatant B and mixed, and then stood in a -20°C refrigerator for 3 hours to obtain crude RNA; the crude RNA was centrifuged at 12000×g and 4°C for 20 minutes. min, after the centrifugation is completed, the supernatant is poured out by tilting the centrifuge tube to obtain a centrifuge tube containing a white RNA precipitate; 75% (v / v) ethanol is prepared with RNase-free water; after pouring 1 mL of 75% ethanol into the centrifuge tube containing the white RNA precipitate, the precipitate is first washed by turning it upside down, and then centrifuged at 12000×g and 4°C for 10 min. After the centrifugation is completed, the supernatant is poured out by tilting the centrifuge tube, and then centrifuged briefly to obtain a centrifuge tube containing the washed RNA precipitate; the excess liquid in the centrifuge tube containing the washed RNA precipitate is discarded with a pipette, the centrifuge tube is turned upside down, and the precipitate is dried to obtain a centrifuge tube containing a dry RNA precipitate; 30 μL of RNase-free water is added to the centrifuge tube containing the dry RNA precipitate to dissolve the precipitate to obtain a lysate; the RNA concentration of the lysate is determined (the sample needs to be vortexed and briefly centrifuged before determining the RNA concentration), and the lysate is adjusted to an RNA concentration of 1000 ng / μL using RNase-free water to obtain a sample to be tested;

[0133] Reverse transcription: prepare the reverse transcription reaction system according to Table 7; add the reverse transcription reaction system to the centrifuge tube, vortex mix, and place in a PCR instrument for reverse transcription reaction (reverse transcription reaction program: 42°C, 2 min; 37°C, 15 min; 85°C, 5 s; 4°C, storage), to obtain a cDNA solution;

[0134] qPCR detection: 18S was used as the internal reference and double-stranded DNA dye (SYBR Geen) was used for quantitative detection; the qPCR reaction system was configured according to Table 8; the qPCR reaction system was added to the microtube, vortexed and mixed, and then placed in the LC480 fluorescence quantitative PCR instrument for PCR reaction (PCR reaction program: 95°C for 5 minutes, 95°C for 30 seconds for 40 cycles, 6°C for 30 seconds, and 72°C for 1 minute; the primers used for qPCR are shown in Table 4).

[0135] Depend on Figure 9~Figure 13 The results showed that compared with the control empty plasmid, the recombinant plasmid CMV-RVG-siR EGFR+VEGFR+RRM2+Serpinh1+anti-214 The obtained exosomes can significantly reduce the expression of EGFR, VEGFR, RRM2, Serpinh1 protein and mRNA in U87 cells. This result shows that the exosomes extracted from mouse plasma have a relatively high interference efficiency on the target gene.

[0136] Table 5 Enzyme digestion system

[0137]

[0138] Table 6 Connection system

[0139]

[0140] Table 7 Reverse transcription reaction system

[0141]

[0142] Table 8 qPCR reaction system

[0143]

[0144] Experimental Example 4: The therapeutic effect of gene circuits in mice

[0145] The specific process is as follows:

[0146] U87-luc cells (purchased from Shanghai Institute of Cell Biology) were cultured at 1×10 6The injection volume of U87 cells (solvent is 5 μL high glucose medium) was implanted into the brain of nude mice (purchased from Jicui Yaokang) (the implantation position is relative to the following coordinates of the anterior fontanelle: 0.5 mm posterior, 2.5 mm lateral and 3.5 mm intracerebral) to construct a glioblastoma xenograft model; 7 days after implantation of U87 cells, the size of the tumor was detected by in vivo imaging to ensure the successful formation of glioblastoma in the brain. The nude mice carrying glioblastoma were randomly divided into 3 groups, namely blank group (n=10), control group (n=10) and treatment group (n=10). The mice in the treatment group were injected with recombinant plasmid CMV-RVG-siR at a dose of 5 mg / kg through the tail vein. EGFR+VEGFR+RRM2+Serpinh1+anti-214 The control group mice were injected with an equal dose of control empty plasmid (solvent was 200 μL PBS buffer) through the tail vein, and the blank group mice were injected with an equal volume of PBS buffer through the tail vein, once every two days, for a total of 14 injections; during the plasmid injection period, mice were imaged once every seven days to detect the size of the tumor. The test results are shown in Figure 14~Figure 15 .

[0147] Depend on Figure 14~Figure 15 The results showed that compared with the control empty plasmid, the recombinant plasmid CMV-RVG-siR EGFR+VEGFR+RRM2+Serpinh1+anti-214 Afterwards, in vivo imaging of small animals on the 28th day showed that the fluorescence intensity of the intracranial tumors in the treated mice was significantly lower than that in the control group (reduced by about 48%). This result shows that the gene circuit can effectively inhibit the deterioration of intracranial tumors in mice.

[0148] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A siRNA targeting glioblastoma, characterized in that: The antisense strand of the siRNA can specifically bind to the target nucleic acid through complementary base pairing to trigger degradation of the target nucleic acid; the target nucleic acid is a gene encoding heat shock protein 47; the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

10.

2. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule comprises an shRNA connection fragment; the shRNA connection fragment comprises an shRNA corresponding to an siRNA targeting an mRNA encoding epidermal growth factor receptor, an shRNA corresponding to an siRNA targeting an mRNA encoding vascular endothelial growth factor receptor, an shRNA corresponding to an siRNA targeting an mRNA encoding ribonucleotide reductase subunit M2, an shRNA corresponding to an siRNA targeting an mRNA encoding heat shock protein 47, and an shRNA corresponding to an siRNA targeting miR-214; The shRNA corresponding to the siRNA targeting the mRNA encoding heat shock protein 47 comprises the sense strand of the siRNA of claim 1 and the antisense strand of the siRNA of claim 1; The shRNA corresponding to the siRNA targeting the mRNA encoding the ribonucleotide reductase subunit M2 comprises a sense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.4 and an antisense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.9; The shRNA corresponding to the siRNA targeting the mRNA encoding epidermal growth factor receptor comprises a sense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.1 and an antisense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.6; The shRNA corresponding to the siRNA targeting miR-214 comprises a sense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.2 and an antisense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.7; The shRNA corresponding to the siRNA targeting the mRNA encoding the vascular endothelial growth factor receptor comprises a sense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.3 and an antisense strand of the siRNA having a nucleotide sequence as shown in SEQ ID NO.8; The sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

3. A recombinant plasmid, characterized in that: The recombinant plasmid expresses the siRNA according to claim 1; or, the recombinant plasmid carries the recombinant nucleic acid molecule according to claim 2.

4. An engineered cell, characterized in that: The genome of the engineered cell is integrated with the siRNA of claim 1; or, the genome of the engineered cell is integrated with the recombinant nucleic acid molecule of claim 2; or, the engineered cell carries the recombinant plasmid of claim 3.

5. A small extracellular vesicle, characterized in that The small extracellular vesicles encapsulate the siRNA according to claim 1; or, the small extracellular vesicles encapsulate the recombinant nucleic acid molecule according to claim 2; or, the small extracellular vesicles encapsulate the recombinant plasmid according to claim 3.

6. Use of the siRNA according to claim 1, the recombinant nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, the engineered cell according to claim 4, or the small extracellular vesicle according to claim 5 in the preparation of a drug for preventing and / or treating glioblastoma.

7. A drug for preventing and / or treating glioblastoma, characterized in that: The ingredients of the drug include an inhibitor; the inhibitor includes the siRNA described in claim 1 or the recombinant nucleic acid molecule described in claim 2 or the recombinant plasmid described in claim 3 or the engineered cell described in claim 4 or the small extracellular vesicle described in claim 5.

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